FXIII detection for verifying serum sample and sample size and for detecting dilution

ABSTRACT

Analyses of serum samples for the presence and amount of either of the two subunits of human Factor XIII protein are used as a means of eliminating a significant source of error that arises in the testing of serum and plasma. For serum samples, a negative result of an analysis for the presence of subunit a is a means of verifying that a sample is indeed serum, while a negative or positive result for subunit a serves to distinguish serum (negative) from plasma (positive). A positive result for the presence of subunit b is a means of verifying that the sample is either serum or plasma and not any other biological fluid. A quantitative analysis of subunit b is a means of verifying that the sample is of the intended volume rather than having been reduced in volume due to improper sampling. A quantitative analysis of subunit b is also a means of verifying the dilution of a sample of either serum or plasma.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention resides in the fields of quality control for clinicallaboratory test procedures and instrumentation, and of human Factor XIIIprotein and its uses.

2. Description of the Prior Art

The medical community, including practicing physicians, researchers, andclinicians of all types rely on the clinical laboratory for analyticaltesting of biological samples as part of routine physical examinations,and in diagnosing disease and monitoring patient progress and diseaseconditions, as well as similar functions and services. Among the mostcommon biological samples that are analyzed by these laboratories areserum and plasma, although other fluids such as urine and cerebrospinalfluid are often used as well. Many analyses are performed by automatedinstrumentation, and in some cases large numbers of samples are analyzedsimultaneously. Whether the tests are performed in this manner or on anindividual basis by a laboratory technician, there are numerous sourcesof error that can produce spurious results.

The error that arises falls within two general classes—(1) spurious testresults due to lapses in standard operating procedures and instrumentmalfunctions, and (2) analytic error. Some of the most common errors ofthe first class are those due to inaccurate mathematical correction forspecimen dilution, misinterpretation of instrument codes, and instrumentsampling errors such as bubbles in sample wells or transfer tubing orother malfunctions that result in samples whose volumes are less thanstandard. Of the second class, the most common type of error is thatcaused by calibration drift. This invention addresses errors of thefirst class. In proficiency testing, these errors have been shown toaccount for 300 false test results per one million assays. A review ofthe causes of laboratory errors is reported by Jenny, R. W., et al.,“Causes of Unsatisfactory Performance in Proficiency Testing,” Clin.Chem. 46(1): 89-99 (2000), who found that inaccurate dilutioncorrections accounted for 21% of spurious test results in proficiencytesting, and missampling in a particular automated instrument such asmight be caused by air bubbles or sample clotting occurred 0.016% of thetime, during use of the instrument in testing for samples from thegeneral population. A further source of error is the use of an incorrectsample type, such as urine, cerebrospinal fluid, or other bodily fluidsinstead of serum, or in coagulation studies the failure to differentiatebetween serum and plasma.

SUMMARY OF THE INVENTION

It has now been discovered that analysis of a biological sample forhuman Factor XIII protein is an effective way of detecting variouserrors of the types discussed above. The two subunits of the protein,hereinafter referred to as “subunit a” or “FXIIIa,” which is recognizedin the art as the activated form of the protein, and “subunit b” or“FXIIIb,” whose function is generally unknown although speculated to bethat of a carrier protein, are analyzed separately in different aspectsof this invention, providing different types of information useful indetecting error. The whole FXIII is a tetramer containing two of each ofthe subunits, and the tetramer as well as the dissociated forms of eachsubunit are present in human plasma. Immmunoassays of human serum,however, detect neither the tetramer nor dissociated subunit a, butinstead detect only subunit b. A small amount of tetramer may be presentin human serum, but if so, the amount is below the detection limit of atypical immunoassay. The terms “serum” and “plasma” as used herein referto human serum and plasma unless otherwise noted.

In one aspect, the present invention resides in a utilization of thefact that subunit b has a narrow physiological range in both plasma andserum and is only rarely deficient. In addition, disease states havesufficiently little effect on the concentration of this subunit.Accordingly, this aspect of the invention resides in a quantitativedetermination of the subunit b in a sample of serum or plasma as anindication of the amount or volume of that sample. If the amount ofsubunit b detected is significantly less than would be present in thesample if the sample were of the intended volume, the determinationserves as an indication of a sampling volume error, i.e., a shortagerelative to the intended volume of the sample.

In another aspect, this invention resides in a method for determiningwhether a sample is serum or plasma, or for verifying that the sample isindeed serum rather than plasma, by analyzing the sample for subunit a,whose presence serves as an indication that plasma constitutes at leastpart, if not all, of the sample composition.

In a third aspect, the invention resides in a method for verifying thata sample that is thought to be serum or plasma is indeed one of thesetwo rather than another biological fluid such as cerebrospinal fluid orurine or non-human serum. This determination is achieved by analyzingthe sample for the presence of subunit b, a positive result indicatingthat the sample is indeed serum or plasma, since subunit b is present inboth serum and plasma and is not present in non-human serum or in otherbiological fluids.

A still further aspect of the invention resides in the analysis of asample of serum or plasma for the quantity or concentration of subunit bin that sample to determine or verify the degree of dilution of thesample. This is achieved by comparing the quantity or concentrationdetected with that of a sample of the same type of fluid but whosedegree of dilution is known.

These and other features and aspects of the invention will be morereadily understood from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph comparing FXIII levels in human serum, urine,cerebrospinal fluid, and non-human sera.

FIG. 2 is a plot showing the distribution of FXIII levels among the seraof 260 human patients.

FIG. 3 is a histogram comparing FXIII levels among serum samples ofdifferent sample sizes.

FIG. 4 is a histogram comparing FXIII levels among serum samples thatare diluted with serum samples that are undiluted.

DETAILED DESCRIPTION OF THE INVENTION AND SPECIFIC EMBODIMENTS

Human blood coagulation factor XIII (FXIII) is a transglutaminase andthe last enzyme in the blood coagulation cascade. Also known asfibrin-stabilizing factor, Laki-Lorand factor, fibrinase, andcrosslinking enzyme, FXIII is responsible for crosslinking the bloodclot by catalyzing the formation of isopeptide bonds between the sidechains of glutamine and lysine. This crosslinking occurs mainly betweenfibrin molecules in the soft clot but also between fibrin andantiplasmin. The factor itself is normally present as the tetramerdescribed above, which is a zymogen that performs its enzymatic functiononly upon activation by thrombin and Ca⁺⁺. Activation occurs throughthrombin cleavage of the Arg 37-Gly 38 peptide bond near the aminoterminus of the a subunits. In the presence of Ca⁺⁺ ions, the b subunitsthen dissociate from the tetramer, unmasking the a subunit. Theactivated form is thus the liberated a subunit, FXIIIa. The b subunit,FXIIIb, is thought to protect or stabilize the a subunit in the tetrameror to regulate the activation of the tetramer in blood plasma. Onceactivated, the a subunit remains attached to fibrin, and the b subunitis released into the serum. Neither the tetramer nor any of theindividual subunits are present in urine or cerebrospinal fluid, ornon-human bodily fluids, that may be used as analytical samples forpurposes of diagnosis or monitoring.

Within the detection limits of conventional immunoassay techniques,therefore, free FXIIIb exists in both serum and plasma (and no otherbodily fluids), while free FXIIIa and the tetramer (containing twochains each of FXIIIa and FXIIIb) exist only in plasma, i.e.:

Plasma: FXIIIa, FXIIIb, and tetramer

Serum: FXIIIb only

The typical concentration of FXIIIb in serum is 21 μg/mL, and anyvariations in patients who are not suffering from congenital FXIIIdeficiency are within the range of approximately 10-40 μg/mL. Theconcentration is independent of the pathophysiological conditions of thebody and unaffected by the coagulation process.

While various methods can be used to detect and/or quantitate either thetetramer or the two subunits, a preferred method of detection isimmunoassay. A particularly convenient class of immunoassays are thosein which the test sample is contacted with a binding reagent that isimmobilized on a solid phase, and one of the steps performed to detectthe presence and/or amount of the analyte is the separation of thespecies in the reaction mixture that have become bound to thesolid-phase binding reagent from species that remain unbound. The solidphase may assume any of a variety of forms, the most prominent examplesof which are the walls of a reaction vessel, such as the individualwells of a multi-well plate, and solid particles that are dispersed inthe assay mixture.

When particles are used, they are preferably microscopic in size, andtherefore referred to as microparticles. The microparticles aregenerally formed of a polymeric material that bears certaincharacteristics that make it useful in immunoassays. One suchcharacteristic is that the matrix be inert to the components of thebiological sample and to the assay reagents other than the assay reagentthat is affixed to the microparticle. Other characteristics are that thematrix be solid and insoluble in the sample and in any other solvents orcarriers used in the assay, and that it be capable of affixing an assayreagent to the microparticle. When the immunoassay is designed such thatfluorescence will be used as the means of detection, the polymericmaterial is preferably one that exhibits minimal autofluorescence.Examples of suitable polymers are polystyrenes, polyesters, polyethers,polyolefins, polyalkylene oxides, polyamides, polyurethanes,polysaccharides, celluloses, and polyisoprenes. Crosslinking is usefulin many polymers for imparting structural integrity and rigidity to themicroparticle. These considerations are also applicable to solid phasesother than microparticles.

The use of particles further offers the ability to classify particlesinto groups that are distinguishable by instrumentation. Multiple assayscan therefore be performed simultaneously, with separate assay resultsindependently determined for each group of particles. Classification ofparticles in this manner can be achieved by embedding identifying agentssuch as fluorochromes or dyes in the body of each particle, usingdifferent such agents, different intensities of such agents, orcombinations of such agents at different ratios among the differentgroups of particles. Flow cytometers that can distinguish amongparticles on these bases are known in the art and available fromcommercial suppliers.

The surface of the solid phase will preferably contain functional groupsfor attachment of the binding member, typically an antibody, that bindsthe analyte. These functional groups can be incorporated into thepolymer structure by conventional means, such as the forming the polymerfrom monomers that contain the functional groups, either as the solemonomer or as a co-monomer. Examples of suitable functional groups areamine groups (—NH₂), ammonium groups (—NH₃ ⁺ or —NR₃ ⁺ where R is analkyl or aryl group), hydroxyl groups (—OH), carboxylic acid groups(—COOH), and isocyanate groups (—NCO). Useful monomers for introducingcarboxylic acid groups into polystyrenes, for example, are acrylic acidand methacrylic acid.

Attachment of the binding member to the solid phase surface can beachieved by electrostatic attraction, specific affinity interaction,hydrophobic interaction, or covalent binding. Covalent binding ispreferred. Linking groups can be used as a means of increasing thedensity of reactive groups on the solid phase surface and decreasingsteric hindrance to achieve maximal range and sensitivity for the assay,or as a means of adding specific types of reactive groups to the solidphase surface to broaden the range of types of assay reagents that canbe affixed to the solid phase. Examples of suitable useful linkinggroups are polylysine, polyaspartic acid, polyglutamic acid andpolyarginine.

In embodiments in which particles are used as the solid phase anddetection is performed by flow cytometry, care should be taken to avoidthe use of particles that emit high autofluorescence since this rendersthem unsuitable for flow cytometry. Particles of low autofluorescencecan be created by standard emulsion polymerization techniques from awide variety of starting monomers. Particles of high porosity andsurface area (i.e., “macroporous” particles), as well as particles witha high percentage of divinylbenzene monomer, should be avoided sincethey tend to exhibit high autofluorescence. Generally, however,microparticles suitable for use in this invention can vary widely insize, and the sizes are not critical to this invention. In most cases,best results will be obtained with microparticle populations whoseparticles range from about 0.3 micrometers to about 100 micrometers,preferably from about 0.5 micrometers to about 20 micrometers, indiameter.

When particles are used as the solid phase, one means of separatingbound from unbound species is to use particles that are made of or thatinclude a magnetically responsive material. Such a material is one thatresponds to a magnetic field. Magnetically responsive materials that canbe used in the practice of this invention include paramagneticmaterials, ferromagnetic materials, ferrimagnetic materials, andmetamagnetic materials. Paramagnetic materials are preferred. Examplesare iron, nickel, and cobalt, as well as metal oxides such as Fe₃O₄,BaFe₁₂O₁₉, CoO, NiO, Mn₂O₃, Cr₂O₃, and CoMnP. The magneticallyresponsive material may constitute the entire particle, but ispreferably only one component of the particle, the remainder being apolymeric material to which the magnetically responsive material isaffixed and which is chemically derivatized as described above to permitattachment of an analyte binding member.

When particles containing magnetically responsive material are used, thequantity of such material in the particle is not critical and can varyover a wide range. The quantity can affect the density of the particle,however, and both the quantity and the particle size can affect the easeof maintaining the particle in suspension. Maintaining suspension servesto promote maximal contact between the liquid and solid phase and tofacilitate flow cytometry. In assays where fluorescence plays a role inthe detection, an excessive quantity of magnetically responsive materialin the particles will also produce autofluorescence at a level highenough to interfere with the assay results. It is therefore preferredthat the concentration of magnetically responsive material be low enoughto minimize any autofluorescence emanating from the material. With theseconsiderations in mind, the magnetically responsive material in aparticle in accordance with this invention preferably ranges from about1% to about 75% by weight of the particle as a whole. A more preferredweight percent range is from about 2% to about 50%, a still morepreferred weight percent range is from about 3% to about 25%, and aneven more preferred weight percent range is from about 5% to about 15%.The magnetically responsive material can be dispersed throughout thepolymer, applied as a coating on the polymer surface or as one of two ormore coatings on the surface, or incorporated or affixed in any othermanner that secures the material in the polymer matrix.

Immunoassays of both the competitive type and the sandwich type can beused. Competitive assays for example can be performed by using solidphase to which molecules of a binding protein (such as an antibody)specific for the analyte are bound. During the assay, the sample and aquantity of labeled analyte, either simultaneously or sequentially, arecontacted with the solid phase. By using a limited number of bindingsites on the solid phase, the assay causes competition between thelabeled analyte and the analyte in the sample for the available bindingsites. After a suitable incubation period, the mixture of liquid andsolid are separated. If particles containing a magnetically responsivematerial are used as the solid phase, separation is achieved by placingthe particles in a magnetic field, causing the particles to adhere tothe walls of the reaction vessel. Otherwise, separation can be achievedby centrifugation or other conventional methods well known among thoseskilled in the use and design of immunoassays. The particles onceseparated are washed to remove any remaining unbound analyte and label.The particles can then be resuspended in a carrier liquid forintroduction into a flow cytometer where the label is detected.

Sandwich assays, also known as immunometric assays, are performed byusing particles (or any solid phase) to which antibody to the analyte isbound. This antibody is termed “capture” antibody. An excess of captureantibody is used relative to the suspected quantity range of the analyteso that all of the analyte binds. The solid phase with capture antibodyattached is placed in contact with the sample, and a second antibody tosame analyte is added, simultaneously or sequentially with the sample.Like the capture antibody, the second antibody is in excess relative tothe analyte, but unlike the capture antibody, the second antibody isconjugated to a detectable label, and may hence be referred to as“label” antibody. The capture and label antibodies bind to differentepitopes on the analyte or are otherwise capable of binding to theanalyte simultaneously in a non-interfering manner. After a suitableincubation period, solid and liquid phases are separated. In the casewhere the solid phase consists of magnetically responsivemicroparticles, the liquid mixture with microparticles suspended thereinis placed under the influence of a magnetic field, causing themicroparticles to adhere to the walls of the reaction vessel, and theliquid phase is removed. The microparticles, still adhering to thevessel wall, are then washed to remove excess label antibody that hasnot become bound to the immobilized analyte, and the microparticles arethen resuspended in a carrier liquid for introduction into a flowcytometer where the amount of label attached to the particles throughthe intervening analyte is detected.

Immunoassays in the practice of this invention can involve the use ofeither monoclonal antibodies or polyclonal antibodies. Antibodies withspecific binding affinity for either of the two subunits (individually)of FXIII and antibodies for the tetramer are available from commercialsuppliers. Such suppliers include Biogenesis Inc., Brentwood, N.H., USA;Affinity Biologics, distributed by U.S. Enzyme Research Laboratories;Calbiochem, San Diego, Calif., USA; The Binding Site, Inc., San Diego,Calif., USA; Biodesign International, Saco, Me., USA; Enzyme ResearchLaboratories, Inc., South Bend, Ind., USA; Fitzgerald IndustriesInternational Inc., Concord, Mass., USA; and Hematologics Inc., Seattle,Wash., USA. In sandwich assays, antibodies can be used in variouscombinations as capture and label antibodies. Thus, to quantify FXIIIa,anti-FXIIIa can be used as the capture antibody and anti-FXIII (i.e.,antibody to the tetramer) as the label antibody. Likewise, anti-FXIII(i.e., antibody to the tetramer) can be used as the capture antibody andanti-FXIIIa as the label antibody. Alternatively, anti-FXIIIa can beused as both capture antibody and label antibody provided that thecapture and label antibodies have specificities to different epitopes onthe FXIIIa molecule. To quantify FXIIIb, anti-FXIIIb can be used as thecapture antibody and anti-FXIII (i.e., antibody to the tetramer) as thelabel antibody. Likewise, anti-FXIII (i.e., antibody to the tetramer)can be used as the capture antibody and anti-FXIIIb as the labelantibody. And likewise further, anti-FXIIIb can be used as both captureantibody and label antibody provided that the capture and labelantibodies have specificities to different epitopes on the FXIIIbmolecule. Other combinations will be readily apparent to those skilledin the art. Either polyclonal or monoclonal antibodies may be used. Whenmonoclonal antibodies are used, they may be either the capture antibody,the label antibody, or both.

Detection of the analyte in the practice of this invention can beaccomplished by any of the wide variety of detection methods that areused or known to be effective in immunological assays. Fluorescence isone example and is readily achieved by the use of fluorophore labels.The wide variety of fluorophores and methods of using them inimmunoassays are well known to those skilled in the immunoassay art, anda wide variety of fluorophores are commercially available. The preferredfluorophores are those that contribute as little autofluorescence aspossible. The fluorophore phycoerythrin is preferred in this regard,since its extinction coefficient and quantum yield are superior to thoseof other fluorophores.

For embodiments of the invention that entail the use of flow cytometry,methods of and instrumentation for flow cytometry are known in the art.Examples of descriptions of flow cytometry instrumentation and methodsin the literature are McHugh, “Flow Microsphere Immunoassay for theQuantitative and Simultaneous Detection of Multiple Soluble Analytes,”Methods in Cell Biology 42, Part B (Academic Press, 1994); McHugh etal., “Microsphere-Based Fluorescence Immunoassays Using Flow CytometryInstrumentation,” Clinical Flow Cytometry, Bauer, K. D., et al., eds.(Baltimore, Md., USA: Williams and Williams, 1993), pp. 535-544; Lindmoet al., “Immunometric Assay Using Mixtures of Two Particle Types ofDifferent Affinity,” J. Immunol. Meth. 126: 183-189 (1990); McHugh,“Flow Cytometry and the Application of Microsphere-Based FluorescenceImmunoassays,” Immunochemica 5: 116 (1991); Horan et al., “Fluid PhaseParticle Fluorescence Analysis: Rheumatoid Factor Specificity Evaluatedby Laser Flow Cytophotometry,” Immunoassays in the Clinical Laboratory,185-189 (Liss 1979); Wilson et al., “A New Microsphere-BasedImmunofluorescence Assay Using Flow Cytometry,” J. Immunol. Meth. 107:225-230 (1988); Fulwyler et al., “Flow Microsphere Immunoassay for theQuantitative and Simultaneous Detection of Multiple Soluble Analytes,”Meth. Cell Biol. 33: 613-629 (1990); Coulter Electronics Inc., UnitedKingdom Patent No. 1,561,042 (published Feb. 13, 1980); Steinkamp etal., Review of Scientific Instruments 44(9): 1301-1310 (1973); andChandler, V. S., et al., U.S. Pat. No. 5,981,180 “Multiplexed Analysisof Clinical Specimens Apparatus and Methods,” issued Nov. 9, 1999(Luminex Corporation).

This invention is useful in both manual procedures and automatedprocedures. The invention is of particular interest in verifying theaccuracy of automated immunoassay analyzers. Examples of suchinstruments are the AxSYM immunoassay analyzer of Abbott LaboratoriesDiagnostics Division, Abbott Park, Ill., USA, and the CODA® immunoassayanalyzer of Bio-Rad Laboratories, Inc., Hercules, Calif., USA.

The methods of this invention can be used in conjunction with anyanalytical procedures that are to be performed on serum or plasmasamples, for analytes indicative of a wide variety of physiological andclinical conditions. The FXIII subunit analysis will verify the accuracyof the sampling volume or the dilution or that the correct sample isbeing analyzed. The two analyses can be performed either simultaneouslyor sequentially.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the invention.

EXAMPLE 1

This example provides the results of sequential immunoassays performedon microparticles to illustrate the ability of the present invention todistinguish plasma from serum by analyzing for the presence ofdissociated subunit a of human Factor XIII protein.

The assays were sandwich-type immunoassays and the microparticles were7.1 μm magnetic microparticles. A portion of the microparticles wascoated with polyclonal anti-FXIIIa₂b₂ antibody and a second portion wascoated with polyclonal anti-FXIIIa antibody (as capture antibodies). Thepolyclonal anti-FXIIIa₂b₂ antibody was specifically reactive toward thetetramer, while the polyclonal anti-FXIIIa antibody was reactive towardboth free and bound a subunit and non-reactive toward free b subunit. Adifferent polyclonal anti-FXIIIa₂b₂ antibody was used as the labelantibody for the tests on both portions. Assays on the first portion ofmicroparticles thus indicated the presence of the tetramer and anydissociated subunits, while assays on the second portion indicated thepresence of dissociated subunit a only.

The microparticles were styrene crosslinked with divinylbenzene andcontaining magnetite. The particle surfaces were carboxylated with acoating layer. Varying amounts of fluorochromes were embedded in eachparticle set to give each group of particles a unique spectral addressor to color-code the particles. The particle coatings were convertedinto active ester form by reaction with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysulfosuccinimide. The active ester-coated particles were thencoupled to antibody at a free amino group on the antibody. Theconversion to active ester form and the coupling of antibody wereperformed according to conventional procedures well known amongimmunologists.

Label antibody was prepared by conjugating antibody to phycoerythrinafter first activating the antibody with succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate and activating thephycoerythrin with sulfosuccinimidyl6-[3′-(2-pyridyldithio)-propionamido]hexanoate/dithiothreitol. Theseactivations and conjugation were likewise performed according toconventional procedures well known among immunologists.

For the first assay (using polyclonal anti-FXIIIa₂b₂ antibody as boththe capture and label antibody), 100 μL of serum was mixed with 100 μLof a particle suspension in wash buffer in which the particleconcentration was 47 μg/mL, while 100 μL of plasma was mixed with 100 μLof a 47 μg/mL particle suspension to form a separate suspension. For thesecond assay (using polyclonal anti-FXIIIa antibody as the captureantibody and polyclonal anti-FXIIIa₂b₂ antibody as the label antibody),serum and plasma were again mixed separately with the particles in thesame proportions to form separate suspensions. In all cases, theparticle/sample suspensions were incubated on a shaker (1,100 rpm) atroom temperature for fifteen minutes. Unbound and contaminating proteinsand immunoglobulins were removed by magnetic separation and washed twicewith wash buffer, with three-minute magnetic separations followed byaspirations between the washes. A final 75-μL volume of wash buffer wasadded to each group for the analyses.

Analyses was performed on a Luminex 100 flow cytometer capable ofmeasuring forward light scatter and particle fluorescence. Theexcitation system of the instrument included a 532 nm reporter laser forexcitation of the phycoerythrin at the microparticle surface, and a 635nm classification laser for excitation of the fluorochomes embedded inthe bulk of the microparticle. The fluorescent emissions that resultedfrom the excitations were discriminated with selective emission filtersand converted into an output signal by a digital signal processor withinthe instrument, the value of the signal indicating the magnitude of thereaction in any particular immunoassay. The instrument was calibrated bycalibration microparticles using appropriate procedures recommended bythe instrument supplier. The values thus calibrated, which areindications of the relative magnitude of the fluorescence intensitiesare referred to herein as “detector units.”

The results expressed in detector units for a series of individualsamples of serum and plasma, as well as the wash buffer alone, arelisted in Table I.

TABLE I Immunoassays on Microparticles: Serum vs. Plasma Detector UnitsPolyclonal Anti-FXIIIa₂b₂ Polyclonal Anti-FXIIIa Antibody as Antibody asBoth Capture and Capture and Polyclonal Anti- Label FXIIIa₂b₂ Antibodyas Label Sample Serum Plasma Serum Plasma A 1906.5 320.8 341.0 4053.5 B1839.5 720.3 419.0 3997.8 C 1549.3 791.5 363.5 4958.0 D 1817.0 750.0287.3 4610.5 E 2136.3 985.0 623.5 4482.0 F 1797.0 758.3 504.0 4415.5 G2233.5 1100.5 679.3 5062.8 H 2138.5 972.3 462.0 5020.5 I 3175.3 2195.5503.3 6044.0 J 2073.8 921.3 468.8 5158.0 K 1933.8 777.0 392.8 4692.5 L1921.0 890.0 509.0 4782.5 Wash Buffer 243.3 249.0

The data in Table I demonstrate that equivalent FXIII signals wereobtained in plasma and serum when both the capture and reporterantibodies had binding affinity to the tetramer (as well as thedissociated subunits), and that no signal was detected in serum when thecapture antibody was specific to the a subunit. The uniqueness of the asubunit to plasma (i.e., the absence of the a subunit in serum) thusdistinguished plasma from serum.

EXAMPLE 2

This example provides the results of ELISAs (enzyme-linked immunosorbentassays) to illustrate the ability of the present invention todistinguish plasma from serum, this time using monoclonal antibodyspecific for the tetramer as the capture antibody in all cases, andpolyclonal antibody specific for subunit a and polyclonal antibodyspecific for subunit b in separate assays as the detection antibody.

The assays were performed on coated microplates, and as in Example 1,two assays were performed on serum and two on plasma. To coat theplates, the capture antibody was diluted {fraction (1/100)} with 50 mMcarbonate buffer, pH 9.6, and added to the microplate wells in volumesof 100 μL per well, followed by incubation overnight at 4° C. per well.The wells were emptied immediately before use and blocking buffer wasadded, followed by four washes with phosphate-buffered saline (PBS) andTween surfactant. Plasma and serum samples were diluted {fraction(1/400)} with PBS and added to the wells. Detection antibody, diluted{fraction (1/1000)} was then added to each well at 100 μL/well, and themicroplates were incubated for sixty minutes at room temperature, washedwith PBS-Tween, incubated again for sixty minutes at room temperature,then rewashed. Peroxidase-conjugated anti-rabbit IgG, diluted {fraction(1/1000)}, was added at 100 μL/well. The peroxidase substratetetramethylbenzidine in an acidic buffer (100 μL) was then added to eachwell, and when the color developed, 100 μL of a stop solution was addedto each well.

The optical density of each well was measured at 450 nm using an ELISAreader. The results are listed in Table II.

TABLE II Immunoassays on Microplates: Serum vs. Plasma Optical Densityat 450 nm Monoclonal Anti-FXIIIa₂b₂ Monoclonal Anti-FXIIIa₂b₂ Antibodyas Capture and Polyclonal Antibody as Capture and Polyclonal Anti-FXIIIaAntibody as Detecting Anti-FXIIIb Antibody as Detecting AntibodyAntibody Sample Plasma Serum Plasma Serum A 1.195 0.677 2.484 2.486 B1.491 0.580 2.873 2.837 C 1.364 0.379 3.062 2.804

These data demonstrate that the a subunit was detected with antibodyspecific to that subunit only in plasma and not in serum, confirmingonce again that the uniqueness of the a subunit to plasma is a means ofdistinguishing plasma from serum.

EXAMPLE 3

This example compares the results of assays for the FXIII tetramer inhuman urine, human cerebrospinal fluid, and serum from various species.

The assays were sandwich-type immunoassays performed on magneticmicroparticles as in Example 1, using monoclonal anti-human FXIIIa₂b₂antibody as the capture antibody and polyclonal anti-human FXIIIa₂b₂antibody conjugated with phycoerythrin as the label antibody. Theparticles, which were 8.0 μm in diameter, were coated with captureantibody in the manner described in Example 1, and antibody-labelconjugates were likewise prepared as described in Example 1. Theparticles were suspended in wash buffer to a concentration of 47 μg/mL,and 100 μL aliquots of the particle suspensions were mixed with 100 μLeach of human serum, cerebrospinal fluid, urine, donkey serum, mouseserum, and goat serum. The resulting particle/sample suspensions wereincubated on a shaker (1,100 rpm) at room temperature for fifteenminutes. Unbound and contaminating proteins were then removed bymagnetic separation. The particles were then washed twice, withthree-minute magnetic separations and aspirations between washes. Theprediluted conjugate (1.8 μg/test) was then added and the suspensionswere incubated again (room temperature, fifteen minutes, 1,100 rpm),followed by magnetic separations and aspirations. Finally, wash buffer(75 μL) was added to prepare the particles for flow cytometry analysis.

Flow cytometry was then performed as in Example 1. The results are shownin bar-graph form in FIG. 1, where the bars, from left to right,represent human urine, human serum, human cerebrospinal fluid, goatserum, donkey serum, and mouse serum. The graph clearly shows that FXIIIwas detected only in human serum, and not in other human bodily fluidsor in the sera of goat, donkey, or mouse.

EXAMPLE 4

This example illustrates how analyses for FXIII in accordance with thisinvention are used to detect short samples, i.e., differences in volumesof human serum samples. The same type of microparticles described abovein Example 3 were used.

In a first set of tests, sample volumes of 50 μL, 10 μL, 3.2 μL, 2.0 μL,and 1 μL were added to microparticle suspensions (100 μL, 47 μg/mL inwash buffer). Incubations, washings and other procedural steps wereperformed as in Example 3, and the results, which were read on theLuminex flow cytometer as detector units, are shown in Table III.

TABLE III Sample Volume Variation Tests: First Set Sample Volume (μL)Detector Units 50.0 3165.0 10.0 2459.8 3.2 2049.6 2.0 1967.3 1.0 1624.0

These results demonstrate that human serum samples that are deficient insize can be detected by analysis of the amount of FXIII.

In a second set of tests, sample volumes of the same size were adjustedto 100 μL with wash buffer before being added to the microparticlesuspension. The procedure was otherwise the same as that used in thefirst set. The results are listed in Table IV.

TABLE IV Sample Volume Variation Tests: Second Set Sample Volume (μL)Prior to Adjustment to 100 μL Detector Units 50.0 3087.8 10.0 2217.2 3.21618.8 2.0 1447.3 1.0 844.5

These results confirm those of the first set.

In a third set, sample volumes of 17 μL, 13 μL, 11 μL, 9 μL, 7 μL, 5 μL,and 3 μL were combined with 390 μL wash buffer, and 100 μL of each wasadded to the microparticle suspension. The procedure was otherwise thesame as that used in the first and second sets, and the results arelisted in Table V.

TABLE V Sample Volume Variation Tests: Third Set Sample Vol- ume (μL)Prior Detector Units to Adding to Sample No. 390 μL 1 2 3 4 5 6 171977.5 1757.0 2102.8 1647.5 2121.8 1724.8 13 1825.3 1624.0 1952.8 1541.51990.3 1696.5 11 1704.5 1482.3 1819.5 1459.5 1876.0 1526.5 9 1573.01332.5 1645.3 1313.0 1783.5 1393.3 7 1414.3 1246.0 1575.0 1179.0 1601.81423.5 5 1211.0 1022.5 1286.3 985.0 1396.8 1202.3 3 921.5 778.5 989.3795.8 1075.0 1076.5 1 465.5 438.0 523.0 467.0 649.5 600.5

These results confirm the results of the first and second sets. In afourth set of tests, 23 serum samples (stored at −70° C.) with volumesof 5 μL, 3 μL, 2 μL, and 1 μL were combined with 295 μL wash buffer, and100 μL of the coated particle suspension (0.28 μg) were added to eachtube. The resulting particle/sample suspensions were incubated on ashaker at 900 rpm at room temperature for fifteen minutes. Unbound andcontaminating proteins and immunoglobulins were removed by magneticseparation. The particles were washed twice with 300 μL wash buffer,with a three-minute magnetic separation and aspiration in betweensuccessive washes. After the final wash, 50 μL of phycoerythrin-labeledanti-FXIII (0.25 μg phycoerythrin per test) was added to each tube. Thetubes are incubated on a skaker at room temperature (15 minutes, 900rpm), followed by a three-minute magnetic separation and aspiration. Theparticles were then washed twice with 300 μL wash buffer, withthree-minute magnetic separations and aspirations between washes. Washbuffer (75 μL) was then added, and the suspension was analyzed on a flowcytometer.

The results are shown in FIG. 3 which shows the cytometer detector unitsand how they are distributed among the various sample sizes. Thehorizontal axis is the detector units and the vertical axis is thenumber of samples, and different sets of vertical bars are used for thevarious sample sizes, filled bars for 5 μL samples, open bars for 1 μLsamples, shaded bars with lines slanting upward to the right for 3 μLsamples, and shaded bars with lines slanting upward to the left for 2 μLsamples. All 1 μL samples are fully distanced from all 5 μL samples,with no overlap between these two sets. This indicates that the presenceof samples that are only 20% of the proper sample volume can beconsistently distinguished from those that are full volume.

EXAMPLE 5

This example illustrates how analyses for FXIII in accordance with thisinvention are used to identify human serum samples that have beendiluted. The same type of microparticles described above in Example 4were used.

In a first set of tests, serum was used in dilutions of {fraction(1/10)}, {fraction (1/20)}, {fraction (1/40)}, {fraction (1/80)}, and1/160 with wash buffer. Each dilution was mixed with a coatedmicroparticle suspension as in the preceding examples. Magneticseparation was then performed immediately (without incubation) to removecontaminating proteins and immunoglobulins. After subsequent washes andincubation with labeled antibody, the microparticles were analyzed onthe Luminex flow cytometer, and the results are shown in Table VI.

TABLE VI Sample Dilution Tests: First Set Dilution Detector Units Neat21319.5 1/10 8576 1/20 2304 1/40 1688.5 1/80 802  1/160 484.5

In a second set of tests, serum was used in dilutions of {fraction(1/40)}, {fraction (1/80)}, {fraction (1/160)}, {fraction (1/320)},{fraction (1/640)}, {fraction (1/1280)}, and {fraction (1/2560)} withwash buffer. Each dilution was mixed with a coated microparticlesuspension as in the preceding examples. Unlike the first set of tests,the particle/sample suspensions in this second set were incubated (roomtemperature, ten minutes, 1,100 rpm) prior to separation. Contaminatingproteins and immunoglobulins were then removed by magnetic separation.After subsequent washes and incubation with label antibody, themicroparticles were analyzed on the Luminex flow cytometer, and theresults are shown in Table VII.

TABLE VII Relative Fluorescence Intensity Dilution Detector Units 1/40 18253.5 1/80  11831 1/160 7052.5 1/320 3933.5 1/640 2338  1/1280 1377.5 1/2560 802.5 Wash Buffer 219.5

The data in Tables VI and VII demonstrate that all dilutions weredistinguishable from each other.

In a third set of tests, 23 serum samples, 100 μL each in volume, wereused, including some that were neat (undiluted) and some that werediluted {fraction (1/10)} with wash buffer. To each sample was added acoated microparticle suspension amounting to 0.26 μg of particles persample, and the samples were separated on a magnetic plate for 3minutes, then aspirated and washed twice with 300 μL wash buffer. Afterthe final wash, labeled antibody (50 μL) was added to each sample. Thesamples were then mixed on a shaker for ten minutes (900 rpm, 37° C.),and then separated on a magnetic plate for 3 minutes. The supernatantwas aspirated and the particles were washed twice with 300 μL washbuffer. Further wash buffer (75 μL) was then added to each sample toprepare the samples for the flow cytometer. Analysis on the flowcytometer proceeded, and the results are shown in the histogram of FIG.4, which shows the distribution of detector units among the neat anddiluted samples. The horizontal axis is the number of detector units andthe vertical axis is the number of samples. Filled bars are used for thediluted samples and open bars for the neat (undiluted) samples. All ofthe diluted samples are fully distanced from the neat samples, with nooverlap between these two sets. This confirms that samples that arediluted by {fraction (1/10)} can be consistently distinguished fromthose that are undiluted.

EXAMPLE 6

This example illustrates the low degree of variation of FXIII among theserum samples of different human patients. Samples from 260 differentindividuals were tested, using the same procedures as Example 3. Thedistribution of results is shown in order of increasing detector unitsin FIG. 2, which indicates that there was little variation among thelevels of FXIII throughout the samples.

The foregoing descriptions are offered primarily for purposes ofillustration. Further modifications and alternatives of the materialsand procedures expressed that are still within the scope of thisinvention above will be readily apparent to those skilled in the art.

What is claimed is:
 1. A method for verifying that an analytical testsample to be analyzed for the presence or absence of a biologicalcondition is a sample of human serum and does not contain human plasma,said method comprising analyzing said sample to detect the presencetherein of the a subunit of human Factor XIII protein, any a subunitthus detected being an indication of the presence of human plasma insaid sample.
 2. A method in accordance with claim 1 in which thepresence of said a subunit is determined by immunoassay.
 3. A method inaccordance with claim 2 in which said immunoassay comprises contactingsaid sample with a capture antibody that has specific binding affinityfor said a subunit and substantially no binding affinity toward the bsubunit of human Factor XIII protein, and detecting said a subunit thuscaptured.
 4. A method in accordance with claim 2 in which saidimmunoassay comprises (i) contacting said sample with a solid phase towhich is bound a capture antibody that has specific binding affinity forsaid a subunit and substantially no binding affinity toward the bsubunit of said human Factor XIII protein, and (ii) determining whetherany binding of said a subunit to said solid phase through said captureantibody has occurred.
 5. A method in accordance with claim 4 in whichsaid solid phase is a population of particles, and (ii) comprisesdetecting said particles to which said a subunit is bound by flowcytometry.
 6. A method in accordance with claim 2 in which saidimmunoassay comprises contacting said sample with a capture antibodythat has binding affinity for said a subunit, and detecting said asubunit thus captured with a detector antibody that has specific bindingaffinity for said a subunit and substantially no binding affinity towardthe b subunit of human Factor XIII protein.
 7. A method in accordancewith claim 2 in which said immunoassay comprises (i) contacting saidsample with a solid phase to which is bound a capture antibody that hasbinding affinity for said a subunit, and (ii) determining whether anybinding of said a subunit to said solid phase through said captureantibody has occurred by detecting any a subunit thus bound with adetector antibody that has specific binding affinity for said a subunitand substantially no binding affinity toward the b subunit of said humanFactor XIII protein.
 8. A method in accordance with claim 7 in whichsaid solid phase is a population of particles, and (ii) comprisesdetecting said particles by flow cytometry.